Laboratory Criteria for Exclusion and Readmission of Potentially Infectious Persons in Sensitive Settings in the Age of Culture-Independent Diagnostic Tests: Report of a Multidisciplinary Workgroup.

Culture-independent diagnostic tests (CIDTs) are increasingly used for clinical diagnosis of gastrointestinal diseases such as salmonellosis, Shiga toxin-producing E. coli disease, and shigellosis because of their speed, convenience, and generally high-performance characteristics. These tests are also used to screen potentially infectious asymptomatic persons during outbreak investigations in sensitive settings such as childcare, food service, and healthcare. However, only limited performance data are available for CIDTs used on specimens from asymptomatic persons. The Association of Public Health Laboratories (APHL) and Council of State and Territorial Epidemiologists (CSTE) convened a workgroup to examine the available scientific data to inform interim decision-making related to exclusion and readmission criteria for potentially infectious persons in sensitive settings, the risks and benefits of different testing strategies, and to identify knowledge gaps for further research. This is the report on the Workgroup findings.

Sugar Modification of Wall Teichoic Acids Determines Serotype-Dependent Strong Biofilm Production in Listeria monocytogenes.

Biofilm production is responsible for persistent food contamination by Listeria monocytogenes, threatening food safety and public health. Human infection and food contamination with L. monocytogenes are caused primarily by serotypes 1/2a, 1/2b, and 4b. However, the association of biofilm production with phylogenic lineage and serotype has not yet been fully understood. In this study, we measured the levels of biofilm production in 98 clinical strains of L. monocytogenes at 37°C, 25°C, and 4°C. The phylogenetic clusters grouped by core genome multilocus sequence typing (cgMLST) exhibited association between biofilm production and phylogenetic lineage and serotype. Whereas clusters 1 and 3 consisting of serotype 4b strains exhibited weak biofilm production, clusters 2 (serotype 1/2b) and 4 (serotype 1/2a) were composed of strong biofilm formers. Particularly, cluster 2 (serotype 1/2b) strains exhibited the highest levels of biofilm production at 37°C, and the levels of biofilm production of cluster 4 (serotype 1/2a) strains were significantly elevated at all tested temperatures. Pan-genome analysis identified 22 genes unique to strong biofilm producers, most of which are related to the synthesis and modification of teichoic acids. Notably, a knockout mutation of the rml genes related to the modification of wall teichoic acids with l-rhamnose, which is specific to serogroup 1/2, significantly reduced the level of biofilm production by preventing biofilm maturation. Here, the results of our study show that biofilm production in L. monocytogenes is related to phylogeny and serotype and that the modification of wall teichoic acids with l-rhamnose is responsible for serotype-specific strong biofilm formation in L. monocytogenes. IMPORTANCE Biofilm formation on the surface of foods or food-processing facilities by L. monocytogenes is a serious food safety concern. Here, our data demonstrate that the level of biofilm production differs among serotypes 1/2a, 1/2b, and 4b depending on the temperature. Furthermore, sugar decoration of bacterial cell walls with l-rhamnose is responsible for strong biofilm production in serotypes 1/2a and 1/2b, commonly isolated from foods and listeriosis cases. The findings in this study improve our understanding of the association of biofilm production with phylogenetic lineage and serotype in L. monocytogenes.

Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans.

Campylobacter enterocolitis may lead to post-infection irritable bowel syndrome (PI-IBS) and while some C. jejuni strains are more likely than others to cause human disease, genomic and virulence characteristics promoting PI-IBS development remain uncharacterized. We combined pangenome-wide association studies and phenotypic assays to compare C. jejuni isolates from patients who developed PI-IBS with those who did not. We show that variation in bacterial stress response (Cj0145_phoX), adhesion protein (Cj0628_CapA), and core biosynthetic pathway genes (biotin: Cj0308_bioD; purine: Cj0514_purQ; isoprenoid: Cj0894c_ispH) were associated with PI-IBS development. In vitro assays demonstrated greater adhesion, invasion, IL-8 and TNFα secretion on colonocytes with PI-IBS compared to PI-no-IBS strains. A risk-score for PI-IBS development was generated using 22 genomic markers, four of which were from Cj1631c, a putative heme oxidase gene linked to virulence. Our finding that specific Campylobacter genotypes confer greater in vitro virulence and increased risk of PI-IBS has potential to improve understanding of the complex host-pathogen interactions underlying this condition.

Evidence of False Positivity for Vibrio Species Tested by Gastrointestinal Multiplex PCR Panels, Minnesota, 2016-2018.

BACKGROUND: Syndromic gastrointestinal multiplex polymerase chain reaction (PCR) panels (GMPPs) are used by an increasing number of clinical laboratories to identify enteric pathogens. Vibrio species are included on GMPPs, but because of the low prevalence of vibriosis, performance characteristics for these panels have been difficult to measure. METHODS: All Vibrio spp. cases identified by GMPPs in Minnesota during 2016-2018 (n = 100) were assessed to identify differences between culture-confirmed cases and those that were PCR-positive only. RESULTS: Overall, 47% of cases had Vibrio species recovered by culture. Two GMPPs were used in Minnesota, Verigene EPT and FilmArray GIP, and the recovery rate of Vibrio spp. was significantly different between these platforms (Verigene EPT 63%, compared with FilmArray GIP 28%). No distinct seasonality was identified among GMPP-positive, culture-negative cases, whereas culture-confirmed case incidence peaked during July and August. Among cases with no other pathogen detected by the GMPP, confirmed cases reported a lower rate of bloody diarrhea (odds ratio [OR], 0.7; P = .004) and were less likely to have a symptom duration >14 days (OR, 0.3; P = .04). Confirmed cases were also more likely to include reports of consuming food items typically associated with Vibrio spp. infection or to have another likely source of infection (eg, international travel or contact with an untreated body of fresh or salt water or marine life; OR, 9.6; P = .001). CONCLUSIONS: The combined findings indicate that cases identified by GMPP that did not have culture confirmation were less likely to include symptoms or exposures consistent with vibriosis. These findings emphasize the need for improvements to testing platform specificity and the importance of combining clinical and exposure information when diagnosing an infection. This study underscores the importance of maintaining the ability to culture Vibrio species to aid in accurate diagnoses.

Characteristics and Risk Factors of Post-Infection Irritable Bowel Syndrome After Campylobacter Enteritis.

BACKGROUND & AIMS: Campylobacter is the leading cause of bacterial gastroenteritis in the United States. We investigated the prevalence of postinfection irritable bowel syndrome (PI-IBS) in a cohort with culture-confirmed Campylobacter cases; risk factors for PI-IBS based on clinical factors; and shifts in IBS patterns postinfection in patients with pre-existing IBS. METHODS: The Minnesota Department of Health collects data on symptoms and exposures upon notification of Campylobacter cases. From 2011 through 2019, we sent surveys (the Rome III and IBS symptom severity surveys) to 3586 patients 6 to 9 months after Campylobacter infection. The prevalence of PI-IBS was estimated and risk factors were assessed using multivariable logistic regression. RESULTS: There were 1667 responders to the survey, 249 of whom had pre-existing IBS. Of the 1418 responders without pre-existing IBS, 301 (21%) subsequently developed IBS. Most of these individuals had IBS-mixed (54%), followed by IBS-diarrhea (38%), and IBS-constipation (6%). The mean IBS symptom severity score was 218 (indicating moderate severity). Female sex, younger age, bloody stools, abdominal cramps, and hospitalization during acute enteritis were associated with increased risk, whereas fever was protective for the development of PI-IBS. Antibiotic use and exposure patterns were similar between PI-IBS and control groups. Among patients with IBS-mixed or IBS-diarrhea before infection, 78% retained their subtypes after infection. In contrast, only 50% of patients with IBS-constipation retained that subtype after infection, whereas 40% transitioned to IBS-mixed. Of patients with pre-existing IBS, 38% had increased frequency of abdominal pain after Campylobacter infection. CONCLUSIONS: In a cohort of patients with Campylobacter infection in Minnesota, 21% developed PI-IBS; most cases reported mixed IBS or diarrhea of moderate severity. Demographic and clinical factors during acute enterocolitis are associated with PI-IBS development. Campylobacter infection also can result in a switch of a pre-existing IBS phenotype.

Genetic characterization of mumps viruses associated with the resurgence of mumps in the United States: 2015-2017.

Despite high coverage with measles, mumps, and rubella vaccine in the United States, outbreaks of mumps occur in close contact settings such as schools, colleges, and camps. Starting in late 2015, outbreaks were reported from several universities, and by the end of 2017, greater than 13,800 cases had been reported nation-wide. In 2013, the CDC and the Association of Public Health Laboratories contracted four Vaccine Preventable Diseases Reference Centers (VPD-RCs) to perform real-time reverse transcription PCR (RT-qPCR) to detect mumps RNA in clinical samples and to determine the genotype. Twelve genotypes of mumps virus are currently recognized by the World Health Organization, and the standard protocol for genotyping requires sequencing the entire gene coding for the small hydrophobic (SH) protein. Phylogenetic analysis of the 1862 mumps samples genotyped from 2015 through 2017 showed that the overall diversity of genotypes detected was low. Only 0.8 % of the sequences were identified as genotypes C, H, J, or K, and 0.5 % were identified as vaccine strains in genotypes A or N, while most sequences (98.7 %) were genotype G. The majority of the genotype G sequences could be included into one of two large groups with identical SH sequences. Within genotype G, a small number of phylogenetically significant outlier sequences were associated with epidemiologically distinct chains of transmission. These results demonstrate that molecular and epidemiologic data can be used to track transmission pathways of mumps virus; however, the limited diversity of the SH sequences may be insufficient for resolving transmission in all outbreaks.

PulseNet and the Changing Paradigm of Laboratory-Based Surveillance for Foodborne Diseases.

PulseNet, the National Molecular Subtyping Network for Foodborne Disease Surveillance, was established in 1996 through a collaboration with the Centers for Disease Control and Prevention; the US Department of Agriculture, Food Safety and Inspection Service; the US Food and Drug Administration; 4 state public health laboratories; and the Association of Public Health Laboratories. The network has since expanded to include 83 state, local, and food regulatory public health laboratories. In 2016, PulseNet was estimated to be helping prevent an estimated 270 000 foodborne illnesses annually. PulseNet is undergoing a transformation toward whole-genome sequencing (WGS), which provides better discriminatory power and precision than pulsed-field gel electrophoresis (PFGE). WGS improves the detection of outbreak clusters and could replace many traditional reference identification and characterization methods. This article highlights the contributions made by public health laboratories in transforming PulseNet's surveillance and describes how the transformation is changing local and national surveillance practices. Our data show that WGS is better at identifying clusters than PFGE, especially for clonal organisms such as Salmonella Enteritidis. The need to develop prioritization schemes for cluster follow-up and additional resources for both public health laboratory and epidemiology departments will be critical as PulseNet implements WGS for foodborne disease surveillance in the United States.

Prevalence and Molecular Characteristics of Clostridium difficile in Retail Meats, Food-Producing and Companion Animals, and Humans in Minnesota.

Community-associated Clostridium difficile infection (CA-CDI) now accounts for approximately 50% of CDI cases in central Minnesota; animals and meat products are potential sources. From November 2011 to July 2013, we cultured retail meat products and fecal samples from food-producing and companion animals in central Minnesota for C. difficile by using standard methods. The resulting 51 C. difficile isolates, plus 30 archived local veterinary C. difficile isolates and 208 human CA-CDI case isolates from central Minnesota (from 2012) from the Minnesota Department of Health, were characterized molecularly, and source groups were compared using discriminant analysis. C. difficile was recovered from 0 (0%) of 342 retail meat samples and 51 (9%) of 559 animal fecal samples. Overall, the 81 animal source isolates and 208 human source isolates were highly diverse genetically. Molecular traits segregated extensively in relation to animal versus human origin. Discriminant analysis classified 95% of isolates correctly by source group; only five (2.5%) human source isolates were classified as animal source. These data do not support meat products or food-producing and companion animals as important sources of CA-CDI in the central Minnesota study region.

Draft Genome Sequences of Four Salmonella enterica subsp. enterica Serovar Enteritidis Strains Implicated in Infections of Avian and Human Hosts.

Salmonella enterica subsp. enterica serovar Enteritidis is a wide-host-range pathogen. Occasionally, it is involved in invasive infections, leading to a high mortality rate. Here, we present the draft genome sequences of four S Enteritidis strains obtained from human and avian hosts that had been involved in bacteremia, gastroenteritis, and primary infections.

Surveillance for and Discovery of Borrelia Species in US Patients Suspected of Tickborne Illness.

Background: Tick-transmitted Borrelia fall into 2 heterogeneous bacterial complexes comprised of multiple species, the relapsing fever (RF) group and the Borrelia burgdorferi sensu lato group, which are the causative agents of Lyme borreliosis (LB), the most common tickborne disease in the Northern Hemisphere. Geographic expansion of LB in the United States and discovery of emerging Borrelia pathogens underscores the importance of surveillance for disease-causing Borrelia. Methods: De-identified clinical specimens, submitted by providers throughout the United States, for patients suspected of LB, anaplasmosis, ehrlichiosis, or babesiosis were screened using a Borrelia genus-level TaqMan polymerase chain reaction (PCR). Borrelia species and sequence types (STs) were characterized by multilocus sequence typing (MLST) utilizing next-generation sequencing. Results: Among 7292 specimens tested, 5 Borrelia species were identified: 2 causing LB, B. burgdorferi (n = 25) and B. mayonii (n = 9), and 3 RF borreliae, B. hermsii (n = 1), B. miyamotoi (n = 8), and Candidatus B. johnsonii (n = 1), a species previously detected only in the bat tick, Carios kelleyi. ST diversity was greatest for B. burgdorferi-positive specimens, with new STs identified primarily among synovial fluids. Conclusions: These results demonstrate that broad PCR screening followed by MLST is a powerful surveillance tool for uncovering the spectrum of disease-causing Borrelia species, understanding their geographic distribution, and investigating the correlation between B. burgdorferi STs and joint involvement. Detection of Candidatus B. johnsonii in a patient with suspected tickborne disease suggests this species may be a previously undetected cause of illness in humans exposed to bat ticks.

Detection of Influenza C Viruses Among Outpatients and Patients Hospitalized for Severe Acute Respiratory Infection, Minnesota, 2013-2016.

Background: Existing literature suggests that influenza C typically causes mild respiratory tract disease. However, clinical and epidemiological data are limited. Methods: Four outpatient clinics and 3 hospitals submitted clinical data and respiratory specimens through a surveillance network for acute respiratory infection (ARI) from May 2013 through December 2016. Specimens were tested using multitarget nucleic acid amplification for 19-22 respiratory pathogens, including influenza C. Results: Influenza C virus was detected among 59 of 10 202 (0.58%) hospitalized severe ARI cases and 11 of 2282 (0.48%) outpatients. Most detections occurred from December to March, 73% during the 2014-2015 season. Influenza C detections occurred among patients of all ages, with rates being similar between inpatients and outpatients. The highest rate of detection occurred among children aged 6-24 months (1.2%). Among hospitalized cases, 7 required intensive care. Medical comorbidities were reported in 58% of hospitalized cases and all who required intensive care. At least 1 other respiratory pathogen was detected in 40 (66%) cases, most commonly rhinovirus/enterovirus (25%) and respiratory syncytial virus (20%). The hemagglutinin-esterase-fusion gene was sequenced in 37 specimens, and both C/Kanagawa and C/Sao Paulo lineages were detected in inpatients and outpatients. Conclusions: We found seasonal circulation of influenza C with year-to-year variability. Detection was most frequent among young children but occurred in all ages. Some cases that were positive for influenza C, particularly those with comorbid conditions, had severe disease, suggesting a need for further study of the role of influenza C virus in the pathogenesis of respiratory disease.

The Possible Influence of Non-synonymous Point Mutations within the FimA Adhesin of Non-typhoidal Salmonella (NTS) Isolates in the Process of Host Adaptation.

Non-typhoidal Salmonella (NTS) remains a global pathogen that affects a wide range of animal species. We analyzed a large number of NTS isolates of different host origins, including Salmonella Heidelberg (n = 80, avian), S. Dublin (50, bovine), S. Typhimurium var 5- (n = 40, porcine), S. 4,5,12,:i:- (n = 40, porcine), S. Cerro (n = 16, bovine), and S. Montevideo (n = 14, bovine), using virulence profiling of the bcfC, mgtC, ssaC, invE, pefA, stn, sopB, and siiE virulence-associated genes, a biofilm production assay, pulsed field gel electrophoresis, and the full-length sequencing of the fimA (adhesin) and iroN (receptor) genes. We determined a key amino acid substitution, A169 (i.e., threonine changed to alanine at position 169), in the FimA protein that changed ligand affinity of FimA toward N-acetyl-D-glucosamine. This finding clearly indicates the important role of non-synonymous single nucleotide polymorphism (nsSNPs) in adhesin functionality that may impact the host tropism of NTS. This nsSNP was found in S. Heidelberg and S. Cerro isolates. Although this was not the case for the IroN receptor, the phylogeny of this receptor and different host origins of NTS isolates were positively correlated, suggesting existence of specific host immune selective pressures on this unique receptor in S. enterica. We found that pefA, a gene encoding major fimbrial subunit, was the most-segregative virulence factor. It was associated with S. Heidelberg, S. Typhimurium var 5- and S. 4,5,12,:i:- but not with the rest of NTS strains. Further, we observed a significantly higher frequency of non-biofilm producers among NTS strains that do not carry pefA (42.5%) compared to S. Heidelberg (2.5%) and S. Typhimurium var 5- (7.5%) and S. 4,5,12,:i:- (0%). This study provides new insights into the host adaptation of avian and mammalian NTS isolates that are based on the bacterial antigens FimA and IroN as well as the interrelationships between host adaptation, overall genetic relatedness, and virulence potential in these NTS isolates.

Single-nucleotide polymorphism typing analysis for molecular subtyping of Salmonella Tennessee isolates associated with the 2007 nationwide peanut butter outbreak in the United States.

BACKGROUND: In 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S. Tennessee to determine the clonal subtypes of S. Tennessee that were associated with the peanut butter outbreak. METHODS AND RESULTS: One seventy-six S. Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S. Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S. Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S. Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S. Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S. Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases. CONCLUSIONS: The SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

Borrelia mayonii sp. nov., a member of the Borrelia burgdorferi sensu lato complex, detected in patients and ticks in the upper midwestern United States.

Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection.

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S. Enteritidis.

Live Animal Markets in Minnesota: A Potential Source for Emergence of Novel Influenza A Viruses and Interspecies Transmission.

BACKGROUND: Live animal markets have been implicated in transmission of influenza A viruses (IAVs) from animals to people. We sought to characterize IAVs at 2 live animal markets in Minnesota to assess potential routes of occupational exposure and risk for interspecies transmission. METHODS: We implemented surveillance for IAVs among employees, swine, and environment (air and surfaces) during a 12-week period (October 2012-January 2013) at 2 markets epidemiologically associated with persons with swine-origin IAV (variant) infections. Real-time reverse transcription polymerase chain reaction (rRT-PCR), viral culture, and whole-genome sequencing were performed on respiratory and environmental specimens, and serology on sera from employees at beginning and end of surveillance. RESULTS: Nasal swabs from 11 of 17 (65%) employees tested positive for IAVs by rRT-PCR; 7 employees tested positive on multiple occasions and 1 employee reported influenza-like illness. Eleven of 15 (73%) employees had baseline hemagglutination inhibition antibody titers ≥40 to swine-origin IAVs, but only 1 demonstrated a 4-fold titer increase to both swine-origin and pandemic A/Mexico/4108/2009 IAVs. IAVs were isolated from swine (72/84), air (30/45), and pen railings (5/21). Whole-genome sequencing of 122 IAVs isolated from swine and environmental specimens revealed multiple strains and subtype codetections. Multiple gene segment exchanges among and within subtypes were observed, resulting in new genetic constellations and reassortant viruses. Genetic sequence similarities of 99%-100% among IAVs of 1 market customer and swine indicated interspecies transmission. CONCLUSIONS: At markets where swine and persons are in close contact, swine-origin IAVs are prevalent and potentially provide conditions for novel IAV emergence.

USA300 and USA500 clonal lineages of Staphylococcus aureus do not produce a capsular polysaccharide due to conserved mutations in the cap5 locus.

UNLABELLED: The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP(-)). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP(-). Whole-genome sequence analysis of 146 CP(-) USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates. IMPORTANCE: The USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP(-)) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP(+)) phenotype is unlikely in vivo or in vitro since it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP(-) and provide new insight into the evolution of the USA300 and USA500 lineages.

Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens.

The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 µl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 µl raw stool; however, 100 µl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

An outbreak of measles in an undervaccinated community.

Measles is readily spread to susceptible individuals, but is no longer endemic in the United States. In March 2011, measles was confirmed in a Minnesota child without travel abroad. This was the first identified case-patient of an outbreak. An investigation was initiated to determine the source, prevent transmission, and examine measles-mumps-rubella (MMR) vaccine coverage in the affected community. Investigation and response included case-patient follow-up, post-exposure prophylaxis, voluntary isolation and quarantine, and early MMR vaccine for non-immune shelter residents >6 months and <12 months of age. Vaccine coverage was assessed by using immunization information system records. Outreach to the affected community included education and support from public health, health care, and community and spiritual leaders. Twenty-one measles cases were identified. The median age was 12 months (range, 4 months to 51 years) and 14 (67%) were hospitalized (range of stay, 2-7 days). The source was a 30-month-old US-born child of Somali descent infected while visiting Kenya. Measles spread in several settings, and over 3000 individuals were exposed. Sixteen case-patients were unvaccinated; 9 of the 16 were age-eligible: 7 of the 9 had safety concerns and 6 were of Somali descent. MMR vaccine coverage among Somali children declined significantly from 2004 through 2010 starting at 91.1% in 2004 and reaching 54.0% in 2010 (χ(2) for linear trend 553.79; P < .001). This was the largest measles outbreak in Minnesota in 20 years, and aggressive response likely prevented additional transmission. Measles outbreaks can occur if undervaccinated subpopulations exist. Misunderstandings about vaccine safety must be effectively addressed.

Neuraminidase H275Y and hemagglutinin D222G mutations in a fatal case of 2009 pandemic influenza A (H1N1) virus infection.

Oseltamivir-resistant 2009 H1N1 influenza virus infections associated with neuraminidase (NA) H275Y have been identified sporadically. Strains possessing the hemagglutinin (HA) D222G mutation have been detected in small numbers of fatal 2009 H1N1 cases. We report the first clinical description of 2009 H1N1 virus infection with both NA-H275Y and HA-D222G mutations detected by pyrosequencing of bronchioalveolar lavage fluid obtained on symptom day 19. The 59-year-old immunosuppressed patient had multiple conditions conferring higher risk of prolonged viral replication and severe illness and died on symptom day 34. Further investigations are needed to determine the significance of infection with strains possessing NA-H275Y and HA-D222G.